This document discusses various microbiological techniques used to culture and isolate bacteria, including streak plating, Gram staining, and culturing in broth. Streak plating involves spreading bacteria across agar plates using an inoculating loop to isolate single colonies. Gram staining distinguishes between Gram-positive and Gram-negative bacteria based on differences in cell wall structure visible under a microscope. Bacteria can also be cultured in nutrient broth to study growth characteristics. These techniques are used to isolate and identify unknown bacterial species.

1. By- Annu Maurya

2.  The streaking is process by which microorganisms
are spread on the surface of the agar medium by an
inoculating loop making streak marks. The
bacterial colonies are produced along the streak
mark. The plate which is prepared by streaking is
called streak plate. Streak plate method is used for
obtaining pure culture from mixed population.
Streak plate gives well separated and isolated
colonies of bacteria which can easily be isolated
preserved as pure culture. This method can also be
used for the study of various characteristics of
bacteria.

3.  Agar Plates
 Bacterial Sample
 Inoculating Loop
 Spirit Lamp
 Incubator

4.  Preparation of an agar plate
 Sterilization of an inoculating loop by read heat
 Cooling of the loop by jabbing in the agar plate
 Small amount of bacterial sample inoculated on
the agar surface by marking parallel streak marks
 Loop sterilizes again and cooled and streaking
continued from the end point of previous streak
mark
 This process repeated several times
 Incubate the streak plates at 350C for 24 to 48 hrs

5.  Bacterial growth could be observed along the
streak marks and separated colonies on the
later streak marks

6. An agar plate streaked with
microorganisms
isolated from a deep-water
sponge. Individual
colonies may be seen center
right.

8.  Streaking is a technique used in microbiology to isolate
a pure strain from a single species of microorganism, often
bacteria. A microbiological culture can be grown so that
the organism can be identified, studied, or tested. A sterile
cotton swab or inoculation loop is sterilized and dipped in
a broth or patient specimen containing many species of
bacteria.

9.  The loop is then spread across one quadrant of an
agar plate containing a growth medium which has
been sterilized in an autoclave. This introduces a
solution of the bacteria or fungi to a substrate
which provides them nutrients. Choice of which
growth medium to use depends on which
microorganism is being cultured, and which are
being selected for, if any. Growth media are
usually based on agar, a gelatinous substance
derived from seaweed.

10.  The loop is re-sterilized and dragged across the
inoculated quadrant of the streak plate. This is
done to collect some bacteria on the loop. The loop
is spread around another fourth of the plate much
like the previous step. The loop is sterilized and
the procedure is repeated. Each time the loop
gathers fewer and fewer bacteria until it gathers
just one single bacterial cell that can grow into a
colony.

11.  The streak plate is then incubated, usually for 24 to
36 hours, to allow the bacteria to reproduce. At the
end of incubation there should be enough bacteria
to form visible colonies in the areas touched by the
inoculation loop. From these mixed colonies,
single bacterial or fungal species can be identified
based on their morphological (size/shape/colour)
differences, and then sub-cultured to a new media
plate to yield a pure culture for further analysis.

12.  Culture of Bacteria in Nutrient Broth

13.  Nutrient broth medium contain sufficient nutrient
and favorable environment for the growth of
majority of bacteria. Nutrient broth is suitable for
the growth of aerobic, anaerobic, facultative,
oxidative and fermentative bacteria. Broth culture
is used for the enrichment and study of many
biochemical characteristics of bacteria.

14.  Test tube
 Nutrient broth medium (Sterilized)
 Micro-pipette
 Bacterial sample (pond water)
 Spirit lamp
 Incubator

15.  Preparation of broth tube (transfer of 5.0 ml
nutrient broth by micro-pipette)
 Inoculation of broth tube with bacterial sample
 Incubation at 350 C until next class

16.  Bacterial growth was indicated by-
 Cloudiness of the medium
 Pellicle or firm formation on the surface of the
medium
 Sediment formation on the bottom of the tube

17.  Culture of Bacteria in an Agar Plate by
Pour Plate Method

18.  This is a method by which bacteria are cultured
throughout the agar medium. Although the
solidified agar medium restricts the movement of
bacteria, the medium is soft enough for the
bacterial growth inside the medium. This method
is used for the quantitative estimation of bacteria,
isolation of bacteria. Study of colony
characteristics and many biochemical
characteristics of bacteria.

19.  Sterilized Petridis
 Melted agar medium
 Bacterial sample
 Pipette
 Spirit lamp
 Incubator

20.  Transfer of bacterial sample into Petridis (0.5-1.0
ml)
 Pouring of melted agar in the Petridis (12-15 ml)
 Mixing the agar with bacterial sample by rotating
the Petridis
 Place the agar plate on a level surface
 Let the agar to be solidified and then incubate the
plate in an inverted position in
 incubator at 350 C for 24-48 hrs

21.  Bacterial colonies formed throughout the medium
 Colony colour and shape were observed

23.  This is a method by which bacteria are cultured on
the surface of solidified agar medium.
 This method is used for the quantitative estimation
of bacteria, isolation of bacteria, study of colony
characteristics and many biochemical
characteristics of bacteria

24.  Agar plates (previously prepared)
 Bacterial sample
 Pipette
 L-shaped glass rod
 Spirit lamp
 Incubator

25.  Transfer of bacterial sample (0.1 ml) on the agar
surface in the Petridis
 Spreading of bacterial sample with the help of L-
shaped glass rod by pushing it back and forth
while rotating the agar plate
 Spreading continued until drying of the sample on
the agar surface
 Incubation of the agar plate in an inverted position
at 300 C until next class

26.  Clear and well separated bacterial colonies formed
on the agar surface
 Colony colour and shape were observed

28.  The Gram’s staining is the most important staining
procedure used in bacteriology. It is a differential
staining method. It differentiate bacteria into gram
(+) and gram (-). In this procedure bacterial sample
divided into two groups:- first group stain into
purple to violet color while the second group pink
to red. The organism staining violet is called gram
(+) and the organism staining to red is called gram
(-).

29.  Bacterial sample
 Grease free clean glass slide
 Inoculating loop
 Staining reagent
 Slide rack
 Reagent tanks
 Immersion oil
 Microscope
 Spirit lamp
 Distilled water

31.  Solution A: Crystal violet- 20.0g (90% dye
content)
Ethyl alcohol- 20 ml (95%)
 Solution B: Ammonium oxalate- 0.85g
Distilled water- 80 ml
 Mix solution A and solution B

32.  Iodine crystal – 5g
 Potassium iodide – 10g
 Distilled water- 10 ml
 Make the 100 ml stock solution in distilled water
to make the final volume 300 ml.

33.  45% acetone 30 ml
 45% ethanol 70 ml

34.  Prepare a smear:- Put a drop of distilled water or
saline on a non greasy clean glass slide.
Aseptically add a little of the colony for staining.
Mix and spread well in the saline or water. Air dry.
Fix by passing the slide 2-3 times through a spirit
lamp flame.

35.  Place the slide in crystal violet for 60 secs
Wash in tap water
Place in Lugol’s iodine 60 sec
Treatment with Acetone-Alcohol for 2-3 sec.
Wash in tap water.
Place in safranin for 30 sec.

36.  Wash in tap water
Air dry
Observed under oil immersion objective lens on
microscope

37.  a. Gram reaction
 b. Cell shape
 c. Cell arrangement
 d. Spore formation

38. Gram-positive anthrax bacteria
(purple rods) in cerebrospinal fluid
sample. If present, a
Gram-negative bacterial species
would appear pink. (The other cells
are white blood cells).

39. Staphylococcus aureus
(Gram positive)

40. Escherichia coli (Gram
negative)

41.  The proteobacteria are a major group of Gram-
negative bacteria. Other notable groups of Gram-
negative bacteria include the cyanobacteria,
spirochaetes, green sulfur and green nonsulfur
bacteria.
 These also include many medically relevant Gram-
negative cocci, bacilli and many bacteria
associated with nosocomial infections.

42.  In the original bacterial phyla, the Gram-positive
forms made up the phylum Firmicutes, a name
now used for the largest group. It includes many
well-known genera such as Bacillus, Listeria,
Staphylococcus, Streptococcus,
Enterococcus,Diplococcus pneumoniae and
Clostridium. It has also been expanded to include
the Mollicutes, bacteria like Mycoplasma that lack
cell walls and so cannot be stained by Gram, but
are derived from such forms.